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Evaluation of Responses Embryogenic Cycas Revoluta Thumb., from Callus Culture Obtained in Vitro

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Evaluation of Responses Embryogenic Cycas Revoluta Thumb., from Callus Culture Obtained in Vitro Journal of Applied Biotechnology & Bioengineering Research Article Open Access Evaluation of responses embryogenic Cycas revoluta thumb., from callus culture obtained in vitro Summary Volume 6 Issue 4 - 2019 Processes cell differentiation and dedifferentiation are included in the development of 1 2 biotechnology protocols to promote somatic embryogenesis as an alternative to the in vitro Rodríguez de la O JL, Lazarous J Flores VA, propagation of plants, somatic embryos may be an excellent strategy for both propagation Robledo Monterrubio MS,3 Contreras Cruz F3 and conservation of fossil species such as cycads. They were evaluated in vitro with 1Department of Plant Science, Autonomous University Chapingo, different strategies, morphogenic responses associated with obtaining somatic embryos Mexico 2 of C. revolute. calli of megagametophytes, subsequently subcultured in four combinations Thesis Master of Science in Biotechnology, Department of Plant of basic salts of Murashige and Skoog (1962) MS, with the addition of benzyladenine Science, Mexico 3 (BA), and 2, dichlorophenoxyacetic (2,4-D) were used, and kinetin (K), and picloram. Professors Autonomous University Chapingo, Mexico In the results, It was possible to characterize potentially embryogenic callus, evaluating Correspondence: Rodríguez de la O JL, Academy of genetics, the levels of both cellular differentiation, necrosis, texture and color; and increases mass Department of Plant Science, Autonomous University Chapingo, or weight considered to start differentiation or proembryogenic or globular type. Calli MexicoCarr. Fed. Mexico Texcoco Km 38.5 CP 56230, Mexico, were subcultured in a medium containing MS salts, incorporating abscisic acid (ABA) in Email 0, 0.38, 1.13, 3.78 and 5.67 uM doses influenced both the production and maturation of somatic embryos. Embryonic structures, presented a pink coloration characteristic strongly Received: July 10, 2019 | Published: August 12, 2019 associated towards maturity. The effect of combinations of BA, Kin, 2,4-D, GA3 and ANA influenced the development and germination of mature somatic embryos. And the combination of 1.36 mM 2,4-D+4.44 uM BA promoted the appearance of calluses with a compact texture, characteristic related to their embryogenic potential. The purpose of this research in Cycas sp was to contribute to the study of the in vitro morphogenic responses of this group of plants. And somatic embryogenesis, will allow the obtaining and multiplication as well as its preservation of Cycas sp. Gender that is evolutionary very important. Keywords: C. revoluta, cycad, somatic embryogenesis, growth regulators Introduction years and that the use of haploid explants or those that do not require fertilization, can significantly optimize genetic resources, since there Cycads are a group of ancestral dioecious, which appeared in are species such as Encephalartos woodii Sander., of which only the pérmico and evolved from the progymnosperms free spores, exemplary and have male sexual propagation is impossible. C. revolute preceding the Gingkoales and Gnetales, succeeded in developing investigations have sought regeneration from ZE;8,9 leaf midrib and mechanisms of adaptation and survival, so have been called “living epicotyls;10 endosperm11 and megagametophytes; however, the use fossils”. Biologically are very interesting because they represent of megagametophytes as a source of explant organogenics responses an important stage in the evolution of flowering plants and are generated (buds and roots), according Ashmore12 are limited for 1 considered baseline in the evolutionary tree seed plants. Their beauty conservation of genetic resources use due to genetic instability and rarity, has generated a great demand, so we have become perfect inherent to these plants. EPE development from embryogenic cultures target of ends and people dedicated to the black market collectors of in cycads, It remains inefficient since cultures cannot be synchronized exotic species, causing today all species of the order Cycadales are and therefore certain developmental events are difficult to identify. included in Appendix I or II of the Convention on International Trade Necrosing problems are frequent and slow responses due to the nature in Endangered Species of Wild Fauna and Flora (CITES, 2017). C. of this type of plant; Litz et al.,7 have indicated that several species of revolute It is the oldest living cycads and is included in Appendix II embryogenic cultures have grown cycad vigorously and are highly of CITES (2017). The species symbolizing economic importance of morphogenic after 11 years of induction. Growth regulators can 2 order, ornamental and besides, they have been attributed to nutritional promote morphogenesis even if the salt concentration is not adequate. and medicinal properties with clear antibacterial and deleterious However, when it is desired to develop ES, use of regulators depends 3–5 effects against colon cancer and epidermoid. However, due to slow on the stage of the process (induction, proliferation, maturation growth, dioicismo, development, and contradictions in the seed, the or germination). While in cycad it has suggested the use of 2,4-D spread is directed to the use of buds or buds in the basal part of the induction, Embryogenic cultures should be subcultured in the same parent plants limiting genetic variability significantly. Thus, It has formulation of basal medium but lacking growth regulators. Litz et emerged a clear need and unpostponable the use of biotechnological al.,7 they founded that these embryogenic cultures are not receptive tools that enable effective propagation of the species to meet demand, to ABA because of its evolutionary condition in the plant kingdom. and contribute to the conservation and avoid extinction. The ES is However, it is essential to remember that many factors other than the in vitro development of embryos from cell culture are not the growth regulators, influencing embryogenic responses, such as the 6 product of a gametic fusion. In the last 70 years are few reports in species and type of explant. Therefore, the objective of this study vitro cultivation in these plants. Somatic embryogenesis in cycads is was to establish the conditions in vitro that can promote the main 7 relatively low and the best responses were obtained from ZE. The morphogenic responses associated with obtaining somatic embryos of morphogenic potential tissue in cycads has been recognized for many Submit Manuscript | http://medcraveonline.com J Appl Biotechnol Bioeng. 2019;6(4):188‒197. 188 © 2019 la et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially. Copyright: Evaluation of responses embryogenic Cycas revoluta thumb., from callus culture obtained in vitro ©2019 la et al. 189 Cycas revolute Thunb., Which allow to establish a protocol alternative obtaining globular structures or proembriogenics. Embryogenic calli propagation and preservation and contribute to study of these species. were subcultured to promote both their differentiation and embryonic responses using different strategies based, the medium with the Materials and methods inorganic salts of Murashige & Skoog14 MS (1962), by changing the The research was conducted at the Laboratory of Plant Tissue presence or absence of auxiliaries and regulators increase. Culture Department of Plant Science at the Autonomous University Chapingo, Mexico. calli three months old were used obtained from a. Strategy 1: MS salts (1962) with the source of nitrate in vitro culture of C. revolute megagametophytes from a culture diluted to 50%, supplemented with myo-inositol, thiamine, medium with macroelements and salts of B5 medium13 and microcells pyridoxine, folic acid, biotin, mannitol, L-cysteine, PVP MS medium (1962) supplemented with arginine, asparagine and (100, 0.40, 0.5, 0.5, 0.5, 170 , 60 and 70 mg L-1, respectively) glutamine (100, 100 and 400mg L-1, respectively), 6% sucrose and and 4% sucrose. 6 g L-1 gellam-gum. Calli were obtained under the effect of two b. Strategies 2: MS (1962) 100% salts, supplemented with combinations of auxin-cytokine (Table 1). The process for promoting thiamine, myo-inositol, glutamine, arginine, asparagine somatic embryogenesis was evaluated in several experimental stages (0.40, 100, 400, 100 and 100 mg L-1, respectively) and 6% and each strategies., Considered as the induction, maturation and sucrose. germination of embryos obtained. c. Strategy 3: MS (1962) diluted to 50% salts, supplemented with myo-inositol, thiamine, pyridoxine, folic acid, biotin, Table 1 Types and auxin-cytokine concentrations applied to megagametophytes mannitol, PVP (100, 0.40,0.50, 0.50, 0.50, 170 and 70mg of Cycas revolute for callus induction L-1) and sucrose 4%. d. Strategy 4: MS (1962) salts, reducing the source of nitrate Treatment Growth regulators (uM) 50%, supplemented with myo-inositol, thiamine, pyridoxine, 2,4-D picloram Kin BA TDZ folic acid, biotin, mannitol, PVP (100, 0.40,0.50, 0.50, 0.50, 170 and 70mg L -1) and 4% sucrose. one 9.05 - 13.93 - - e. The media were adjusted to pH 5.7±0.1, the effect of three two - 27.75 - 2.13 2.22 different combinations of growth regulators and a control (Table 2) was tested. Promotion stage callus. Mass increases, cell differentiation, and Table 2 Types and concentrations of growth regulators applied to amorphous callus Cycas revolute in proliferation step Growth regulators employees (uM) Strategies Treatment BA 2,4-D Kin picloram 0 - - - - one 1.33 0.45 - - one two 4.44 1.36 - - 3 13.32 4.52 - - 0 - - - - one - 0.45 2.32 - two two - 2.26 4.65 - 3 - 6.79 13.94 - 0 - - - - one - - 0.46 4.14 3 and 4 two - - 2.32 4.14 3 4.65 4.14 Variables evaluated To identify the physical characteristics of callus associated with their embryogenic potential was established a model to determine levels of cellular organization or differentiation, until the formation of globular structures or proembriogenics type, having as variables. 1. Levels cell differentiation (DC), with: 0, no differentiation (callus); 1, minimal differentiation; 2, cell aggregates 3.
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